DNA immunization against chlamydia infection

ABSTRACT

Nucleic acid, including DNA, immunization to generate a protective immune response in a host, including humans, to a major outer membrane protein of a strain of Chlamydia, preferably contains a nucleotide sequence encoding a MOMP or a MOMP fragment that generates antibodies that specifically react with MOMP and a promoter sequence operatively coupled to the first nucleotide sequence for expression of the MOMP in the host. The non-replicating vector may be formulated with a pharmaceutically acceptable carrier for in vivo administration to the host.

REFERENCES TO RELATED APPLICATION

This application is a US National Phase filing of PCT Application No.PCT/CA97/00500 filed Jul. 11, 1997 which claims priority under 35 USC119(e) from U.S. Provisional Application No. 60/021,607 filed Jul. 12,1996.

FIELD OF INVENTION

The present invention relates to immunology and, in particular, toimmunization of hosts using nucleic acid to provide protection againstinfection by Chlamydia.

BACKGROUND OF THE INVENTION

DNA immunization is an approach for generating protective immunityagainst infectious diseases (ref. 1—throughout this application, variousreferences are cited in parentheses to describe more fully the state ofthe art to which this invention pertains. Full bibliographic informationfor each citation is found at the end of the specification, immediatelypreceding the claims. The disclosure of these references are herebyincorporated by reference into the present disclosure). Unlike proteinor peptide based subunit vaccines, DNA immunization provides protectiveimmunity through expression of foreign proteins by host cells, thusallowing the presentation of antigen to the immune system in a mannermore analogous to that which occurs during infection with viruses orintracellular pathogens (ref. 2). Although considerable interest hasbeen generated by this technique, successful immunity has been mostconsistently induced by DNA immunization for viral diseases (ref. 3).Results have been more variable with non-viral pathogens which mayreflect differences in the nature of the pathogens, in the immunizingantigens chosen, and in the routes of immunization (ref. 4). Furtherdevelopment of DNA vaccination will depend on elucidating the underlyingimmunological mechanisms and broadening its application to otherinfectious diseases for which existing strategies of vaccine developmenthave failed.

Chlamydia trachomatis is an obligate intracellular bacterial pathogenwhich usually remains localized to mucosal epithelial surfaces of thehuman host. Chlamydiae are dimorphic bacteria with an extracellularspore-like transmission cell termed the elementary body (EB) and anintracellular replicative cell termed the reticulate body (ref. 5). Froma public health perspective, chlamydial infections are of greatimportance because they are significant causes of infertility, blindnessand are a prevalent co-factor facilitating the transmission of humanimmunodeficiency virus type 1 (ref. 6). Protective immunity to C.trachomatis is effected through cytokines released by Th1-like CD 4lymphocyte responses and by local antibody in mucosal secretions and isbelieved to be primarily directed to the major outer membrane protein(MOMP), which is quantitatively the dominant surface protein on thechlamydial bacterial cell and has a molecular mass of about 40 kDa (ref.19).

Initial efforts in developing a chlamydial vaccine were based onparenteral immunization with the whole bacterial cell. Although thisapproach met with success in human trials, it was limited becauseprotection was short-lived, partial and vaccination may exacerbatedisease during subsequent infection episodes possibly due topathological reactions to certain chlamydial antigens (ref. 8). Morerecent attempts at chlamydial vaccine design have been based on asubunit design using MOMP protein or peptides. These subunit vaccineshave also generally failed, perhaps because the immunogens do not induceprotective cellular and humoral immune responses recalled by nativeepitopes on the organism (ref. 9).

EP 192033 describes the provision of DNA construct for the expression,in vitro, of Chlamydia trachomatis MOMP polypeptides comprising thefollowing operably linked elements:

a transcriptional promoter,

a DNA molecule encoding a C. trachomatis MOMP polypeptide comprising aMOMP polynucleotide at least 27 base pairs in length from a sequenceprovided in Appendix A thereto, and

a transcriptional terminator, wherein at least one of thetranscriptional regulatory elements is not derived from Chlamydiatrachomatis. There is no disclosure or suggestion in this prior art toeffect DNA immunization with any such constructs.

WO 94/26900 describes the provision of hybrid picornaviruses whichexpress chlamydial epitopes from MOMP of Chlamydia trachomatis and whichis capable of inducing antibodies immuno-reactive with at least threedifferent Chlamydia serovars. The hybrid picornavirus preferably is ahybrid polio virus which is attenuated for human administration.

SUMMARY OF THE INVENTION

The present invention is concerned with nucleic acid immunization,specifically DNA immunization, to generate in a host protectiveantibodies to a MOMP of a strain of Chlamydia. DNA immunization inducesa broad spectrum of immune responses including Th1-like CD4 responsesand mucosal immunity.

Accordingly, in one aspect, the present invention provides animmunogenic composition in vivo for in vivo administration to a host forthe generation in the host of a protective immune response to a majorouter membrane protein (MOMP) of a strain of Chlamydia, comprising anon-replicating vector comprising a nucleotide sequence encoding a MOMPor MOMP fragment that generates a MOMP-specific immune response, and apromoter sequence operatively coupled to said nucleotide sequence forexpression of said MOMP in the host; and a pharmaceutically-acceptablecarrier therefor.

The nucleotide sequence may encode a full-length MOMP protein or mayencode a fragment, such as the N-terminal half of MOMP. The nucleotidesequence may encode a MOMP which stimulates a recall immune responsefollowing exposure to wild-type Chlamydia. The promoter may be thecytomegalovirus promoter.

The strain of Chlamydia may be a strain of Chlamydia inducing chlamydialinfection of the lung, including Chlamydia trachomatis or Chlamydiapneumoniae. The non-replicating vector may be plasmid pcDNA3 into whichthe nucleotide sequence is inserted. The immune response which isstimulated may be predominantly a cellular immune response.

In a further aspect of the invention, there is provided as a method ofimmunizing a host against disease caused by infection with a strain ofChlamydia, which comprises administering to said host an effectiveamount of a non-replicating vector comprising a nucleotide sequenceencoding a major outer membrane protein (MOMP) of a strain of Chlamydiaor a MOMP fragment that generates a MOMP-specific immune response, and apromoter sequence operatively coupled to said nucleotide sequence forexpression of said MOMP in the host.

In this aspect of the present invention, the various options andalternatives discussed above may be employed.

The non-replicating vector may be administrated to the host, including ahuman host, in any convenient manner, such as intramuscularly orintranasally. Intranasal administration stimulated the strongest immuneresponse in experiments conducted herein.

The present invention also includes, in an additional aspect thereof,wherein said non-replicating vector comprises plasmid pcDNA3 containingthe promoter sequence and into which the nucleotide sequence is insertedin operative relation to the promoter sequence.

In the additional aspect of the invention, a further aspect of thepresent invention provides a method of producing a vaccine forprotection of a host against disease caused by infection with a strainof Chlamydia, which comprises isolating a nucleotide sequence encoding amajor outer membrane protein (MOMP) of a strain of Chlamydia or a MOMPfragment that generates a MOMP-specific immune response, operativelylinking said nucleotide sequence to at least one control sequence toproduce a non-replicating vector, the control sequence directingexpression of said MOMP when introduced to a host to produce an immuneresponse to said MOMP, and formulating said vector as a vaccine for invivo administration to a host.

Advantages of the present invention, therefore, include a method ofobtaining a protective immune response to infection carried by a strainof Chlamydia by DNA immunization of DNA encoding the major outermembrane protein of a strain of Chlamydia.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates delayed-type hypersensitively (DTH) responsesfollowing immunization. Balb/c mice (four per group) were immunizedintramuscularly (pMOMP IM) or intranasally (pMOMP IN) with plasmid DNAcontaining the coding sequence of the MoPn MOMP gene or with MoPnelementary bodies (EB) at 0,3,6 weeks. The control group was treatedwith the blank plasmid vector (pcDNA3). Fifteen days after the lastimmunization, mice were tested for MoPn-specific DTH response asfollows: 25 μl of heat-inactivated MoPn EB (5×10⁴ IFU) in SPG buffer wasinjected into the right hind footpad and the same volume of SPG bufferwas injected into the left hind footpad. Footpad swelling was measuredat 48 H and 72 H following the injection. The difference between thethickness of the two footpads was used as a measure of the DTH response.Data are shown as the mean±SEM.

FIG. 2, having panels A and B, illustrate protection against MoPninfection with momp gene products following DNA immunization. The Balb/cmice were immunized with (o) pcDNA3 (n=11), () pMOMP intramuscularlly(n=12), (Δ) pMOMP intranasally (n=5) or (▾) MoPn EBs (n=12). Eighteendays after the last immunization, mice were challenged intranasally withinfectious MoPn (1000 IFU). Panel A shows body weight loss. Body weightwas measured daily following infection challenge and each pointrepresents the mean±SEM of the body weight loss. Panel B shows in vivochlamydia clearance. Mice were sacrificed day 10 postinfection andrecovery of infectious MoPn from lung tissue was analyzed byquantitative tissue culture in order to determine the in vivo chlamydialclearance. The data represent mean±SEM of the log₁₀ IFU per lung.

FIG. 3 illustrates detection of serum antibody to MoPn MOMP in DNAimmunized mice by immunoblot analysis. Day 60 pooled sera from miceimmunized with MoPn EBs (Lane A), pMOMP (Lane B), blank pcDNA3 vector(Lane C) or saline (Lane D), were diluted at 1:100 and reacted withpurified MoPn EBs that had been separated in a 10% SDS-polyacrylamidegel and transferred to a nitrocellulose membrane.

FIG. 4, having panels A, B, C and D, compares serum 1 gG subclasses 1gG_(2a) (Panels A and C) with 1 gG, Panels B and D) against recombinantMOMP protein (Panels A and B) or MoPn EBs (Panels C and D) induced byDNA immunization. Mice were non-immunized or immunized intramuscularlywith pMOMP, CTP synthetase DNA (pCTP) or the blank plasmid vector(pcDNA3) at 0,3,6 weeks and pooled sera from each group were collectedtwo weeks following the last immunization (day 10). The data representmean±SEM of the OD value of four duplicates.

FIG. 5, having panels A and B, demonstrates that DNA vaccination withthe MOMP gene enhanced clearance of MoPn infection in the lung. Groupsof Balb/c mice were immunized with pMOMP (n=10), pcDNA3 (n=10) or saline(n=5). Eighteen days after the last immunization, the mice werechallenged intranasally with infectious MoPn (10⁴ IFU). Panel A showsthe body weight of the mice measured daily following challenge infectionuntil the mice were sacrificed at day 10. Each point represents themean±SEM of the body weight change. * represents P<0.05 compared withpcDNA3 treated group. Panel B: the mice were sacrificed at day 10postinfection and the MoPn growth in the lung was analyzed byquantitative tissue culture. The data represent mean±SEM of the Log₁₀IFUper lung. * represents P<0.01 compared with pcDNA3 treated group.

FIG. 6, having panels A and B, shows evaluation of the responses of miceto MoPn intranasal challenge infection. In Panel A, is shown change inbody weight post challenge and in Panel B, is shown the growth of MoPnin lung tissue collected 10 days after challenge. Mice were shamimmunized, □ immunized intraperitoneally with MoPn EBs (when killed ),recovered from prior MoPn lung infection (▾) or immunizedintramuscularly with p½ MOMP(574 ).

FIG. 7 shows the elements and construction of plasmid pcDNA3/MOMP.

GENERAL DESCRIPTION OF THE INVENTION

To illustrate the present invention, plasmid DNA was constructedcontaining the MOMP gene from the C. trachomatis mouse pneumonitisstrain (MoPn), which is a natural murine pathogen, permittingexperimentation to be effected in mice. It is known that primaryinfection in the model induces strong protective immunity toreinfection. For human immunization, a human pathogen strain is used.

Any convenient plasmid vector may be used, such as pcDNA3, a eukaryoticII-selectable expression vector (Invitrogen, San Diego, Calif., USA),containing a Cyotmegalovirus promoter. The MOMP gene may be inserted inthe vector in any convenient manner. The gene may be amplified fromChlamydia trachomatic genomic DNA by PCR using suitable primers and thePCR product cloned into the vector. The MOMP gene-carrying plasmid maybe transferred, such as by electroperation, into E. coli for replicationtherein. Plasmids may be extracted from the E. coli in any convenientmanner.

The plasmid containing the MOMP gene may be administered in anyconvenient manner to the host, such as intramuscularly or intranasally,in conjunction with a pharmaceutically-acceptable carrier. In theexperimentation outlined below, it was found that intranasaladministration of the plasmid DNA elicited the strongest immuneresponse.

The data presented herein and described in detail below demonstratesthat DNA immunization with the C. trachomatis MOMP gene elicits bothcellular and humoral immune responses and produces significantprotective immunity to lung challenge infection with C. trachomatisMoPn. The results are more encouraging than those obtained usingrecombinant MOMP protein or synthetic peptides and suggest that DNAimmunization is an alternative method to deliver a chlamydial subunitimmunogen in order to elicit the requisite protective cellular andhumoral immune responses.

The data presented herein also demonstrate the importance in selectionof an antigen gene for DNA immunization. The antigen gene elicits immuneresponses that are capable of stimulating recall immunity followingexposure to the natural pathogen. In particular, injection of a DNAexpression vector encoding the major outer surface protein (the pMOMP)but not one encoding a cytoplasmic enzyme (CTP synthetase) of C.trachomatis generated significant protective immunity to subsequentchlamydial challenge. The protective immune response appeared to bepredominantly mediated by cellular immunity and not by humoral immunitysince antibodies elicited by DNA vaccination did not bind to native EBs.In addition, MOMP DNA but not CTP synthetase DNA immunization elicitedcellular immunity readily recalled by native EBs as shown by positiveDTH reactions.

In addition, mucosal delivery of MOMP DNA is demonstrated herein to besignificantly more efficient in inducing protective immunity to C.trachomatis infection than intramuscular injection. This may be relevantto the nature of C. trachomatis infection which is essentiallyrestricted to mucosal surfaces and the efficiency of antigenpresentation (ref. 14). The rich population and rapid recruitment ofdendritic cells into the respiratory epithelium of the lung may berelevant to the enhanced efficacy of intranasal DNA immunizationexperiments (ref. 15). The data presented herein represents thedemonstration of a first subunit chlamydial vaccine which engenderssubstantial protective immunity.

Additionally, it may be possible to amplify (and/or canalize) theprotective immune response by co-administration of DNAs that expressimmunoregulatory cytokines in addition to the antigen gene in order toachieve complete immunity (ref. 21) The use of multiple antigen genesfrom chlamydiae may augment the level of protective immunity achieved byDNA vaccination.

A possible concern regarding MOMP DNA immunization stems from theobservation that the MOMP among human C. trachomatis strains is highlypolymorphic (ref. 16) and hence it may be difficult to generate auniversal chlamydial vaccine based on this antigen gene. One way tosolve this problem may be to search for conserved protective epitope(s)within the MOMP molecule. Another, possibly more feasible way, is todesign a multivalent vaccine based on multiple MOMP genes. The latterapproach is justified by the fact that the inferred amino acid sequencesof MOMP among related serovars is relatively conserved and therepertoire of C. trachomatis genevariants appears to be finite (ref.16).

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis and treatment of chlamydial infections.A further non-limiting discussion of such uses is further presentedbelow.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from the MOMP genes and vectors as disclosed herein. Thevaccine elicits an immune response in a subject which includes theproduction of anti-MOMP antibodies. Immunogenic compositions, includingvaccines, containing the nucleic acid may be prepared as injectables, inphysiologically-acceptable liquid solutions or emulsions forpolynucleotide administration. The nucleic acid may be associated withliposomes, such as lecithin liposomes or other liposomes known in theart, as a nucleic acid liposome (for example, as described in WO9324640, ref. 12) or the nucleic acid may be associated with anadjuvant, as described in more detail below. Liposomes comprisingcationic lipids interact spontaneously and rapidly with polyanions, suchas DNA and RNA, resulting in liposome/nucleic acid complexes thatcapture up to 100% of the polynucleotide. In addition, the polycationiccomplexes fuse with cell membranes, resulting in an intracellulardelivery of polynucleotide that bypasses the degradative enzymes of thelysosomal compartment. Published PCT application WO 94/27435 describescompositions for genetic immunization comprising cationic lipids andpolynucleotides. Agents which assist in the cellular uptake of nucleicacid, such as calcium ions, viral proteins and other transfectionfacilitating agents, may advantageously be used.

Polynucleotide immunogenic preparations may also be formulated asmicrocapsules, including biodegradable time-release particles. Thus,U.S. Pat. No. 5,151,264 describes a particulate carrier of aphospholipid/glycolipid/polysaccharide nature that has been termed BioVecteurs Supra Moleculaires (BVSM). The particulate carriers areintended to transport a variety of molecules having biological activityin one of the layers thereof.

U.S. Pat. No. 5,075,109 describes encapsulation of the antigenstrinitrophenylated keyhole limpet hemocyanin and staphylococcalenterotoxin B in 50:50 poly (DL-lactideco-glycolide). Other polymers forencapsulation are suggested, such as poly(glycolide),poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters andpoly(8-hydroxybutyric acid), and polyanhydrides.

Published PCT application WO 91/06282 describes a delivery vehiclecomprising a plurality of bioadhesive microspheres and antigens. Themicrospheres being of starch, gelatin, dextran, collagen or albumin.This delivery vehicle is particularly intended for the uptake of vaccineacross the nasal mucosa. The delivery vehicle may additionally containan absorption enhancer.

The MOMP gene containing non-replicating vectors may be mixed withpharmaceutically acceptable excipients which are compatible therewith.Such excipients may include, water, saline, dextrose, glycerol, ethanol,and combinations thereof. The immunogenic compositions and vaccines mayfurther contain auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessthereof. Immunogenic compositions and vaccines may be administeredparenterally, by injection subcutaneously, intravenously, intradermallyor intramuscularly, possibly following pretreatment of the injectionsite with a local anesthetic. Alternatively, the immunogeniccompositions formed according to the present invention, may beformulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkylene glycols ortriglycerides. Oral formulations may include normally employedincipients, such as, for example, pharmaceutical grades of saccharine,cellulose and magnesium carbonate.

The immunogenic preparations and vaccines are administered in a mannercompatible with the dosage formulation, and in such amount as will betherapeutically effective, protective and immunogenic. The quantity tobe administered depends on the subject to be treated, including, forexample, the capacity of the individual's immune system to synthesizethe MOMP and antibodies thereto, and if needed, to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of about 1 μg to about 1 mg of theMOMP gene-containing vectors. Suitable regimes for initialadministration and booster doses are also variable, but may include aninitial administration followed by subsequent administrations. Thedosage may also depend on the route of administration and will varyaccording to the size of the host. A vaccine which protects against onlyone pathogen is a monovalent vaccine. Vaccines which contain antigenicmaterial of several pathogens are combined vaccines and also belong tothe present invention. Such combined vaccines contain, for example,material from various pathogens or from various strains of the samepathogen, or from combinations of various pathogens.

Immunogenicity can be significantly improved if the vectors areco-administered with adjuvants, commonly used as 0.05 to 0.1 percentsolution in phosphate-buffered saline. Adjuvants enhance theimmunogenicity of an antigen but are not necessarily immunogenicthemselves. Adjuvants may act by retaining the antigen locally near thesite of administration to produce a depot effect facilitating a slow,sustained release of antigen to cells of the immune system. Adjuvantscan also attract cells of the immune system to an antigen depot andstimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years toimprove the host immune responses to, for example, vaccines. Thus,adjuvants have been identified that enhance the immune response toantigens. Some of these adjuvants are toxic, however, and can causeundesirable side-effects, making them unsuitable for use in humans andmany animals. Indeed, only aluminum hydroxide and aluminum phosphate(collectively commonly referred to as alum) are routinely used asadjuvants in human and veterinary vaccines.

A wide range of extrinsic adjuvants and other immunomodulating materialcan provoke potent immune responses to antigens. These include saponinscomplexed to membrane protein antigens to produce immune stimulatingcomplexes (ISCOMS), pluronic polymers with mineral oil, killedmycobacteria in mineral oil, Freund's complete adjuvant, bacterialproducts, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS),as well as monophoryl lipid A, QS 21 and polyphosphazene.

In particular embodiments of the present invention, the non-replicatingvector comprising a first nucleotide sequence encoding a MOMP gene ofChlamydia may be delivered in conjunction with a targeting molecule totarget the vector to selected cells including cells of the immunesystem.

The non-replicating vector may be delivered to the host by a variety ofprocedures, for example, Tang et al. (ref. 17) disclosed thatintroduction of gold microprojectiles coated with DNA encoding bovinegrowth hormone (BGH) into the skin of mice resulted in production ofanti-BGH antibodies in the mice, while Furth et al. (ref. 18) showedthat a jet injector could be used to transfect skin, muscle, fat andmammary tissues of living animals.

2. Immunoassays

The MOMP genes and vectors of the present invention are useful asimmunogens for the generation of anti-MOMP antibodies for use inimmunoassays, including enzyme-linked immunosorbent assays (ELISA), RIAsand other non-enzyme linked antibody binding assays or procedures knownin the art. In ELISA assays, the non-replicating vector first isadministered to a host to generate antibodies specific to the MOMP.These MOMP specific antibodies are immobilized onto a selected surface,for example, a surface capable of binding the antibodies, such as thewells of a polystyrene microtiter plate. After washing to removeincompletely adsorbed antibodies, a nonspecific protein, such as asolution of bovine serum albumin (BSA) that is known to be antigenicallyneutral with regard to the test sample, may be bound to the selectedsurface. This allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This procedure may includediluting the sample with diluents, such as solutions of BSA, bovinegamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. Thesample is then allowed to incubate for from about 2 to 4 hours, attemperatures such as of the order of about 20° to 37° C. Followingincubation, the sample-contacted surface is washed to removenon-immunocomplexed material. The washing procedure may include washingwith a solution, such as PBS/Tween or a borate buffer. Followingformation of specific immunocomplexes between the test sample and thebound MOMP specific antibodies, and subsequent washing, the occurrence,and even amount, of immunocomplex formation may be determined.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitation.

EXAMPLE 1

This Example illustrates the preparation of a plasmid vector containingthe MOMP gene.

pMOMP expression vector was made as follows. The MOMP gene was amplifiedfrom Chlamydia trachomatis mouse pneumonitis (MoPn) strain genomic DNAby polymerase chain reaction (PCR) with a 5′ primer(GGGGTCCGCCACCATGCTGCCTGTGGGGAATCCT) (SEQ ID NO: 1) which includes aBamH1 site, a ribosomal binding site, an initiation codon and theN-terminal sequence of the mature MOMP of MoPn and a 3′ primer(GGGGCTCGAGCTATTAACGGAACTGAGC) (SEQ ID NO: 2) which includes theC-terminal sequence of the MoPn MOMP, a Xhol site and a stop codon. TheDNA sequence of the MOMP leader peptide gene sequence was excluded.After digestion with BamH1 and Xhol, the PCR product was cloned into thepcDNA3 eukaryotic II-selectable expression vector (Invitrogen, SanDiego) with transcription under control of the human cytomegatovirusmajor intermediate early enhancer region (CMV promoter). The MOMPgene-encoding plasmid was transferred by electroporation into E. coliDH5αF which was grown in LB broth containing 100 μg/ml of ampicillin.The plasmids was extracted by Wizard™ Plus Maxiprep DNA purificationsystem (Promega, Madison). The sequence of the recombinant MOMP gene wasverified by PCR direct sequence analysis, as described (ref. 20).Purified plasmid DNA was dissolved in saline at a concentration of 1mg/ml. The DNA concentration was determined by a DU-62 spectrophotometer(Beckman, Fullerton, Calif.) at 260 nm and the size of the plasmid wascompared with DNA standards in ethidium bromide-stained agarose gel.

The MOMP gene containing plasmid, pcDNA3/MOMP is illustrated in FIG. 7.

EXAMPLE 2

This Example illustrates DNA immunization of mice and the results of DTHtesting.

A model of murine pneumonia induced by the C. trachomatis mousepneumonitis strain [MoPn] was used (ref. 11). Unlike most strains of C.trachomatis which are restricted to producing infection and disease inhumans, MoPn is a natural murine pathogen. It has previously beendemonstrated that primary infection in this model induces strongprotective immunity to reinfection. In addition, clearance of infectionis related to CD4 Th1 lymphocyte responses and is dependent on MHC classII antigen presentation (ref. 11).

For experimental design, groups of 4 to 5 week old female Balb/c mice (5to 13 per group) were immunized intramuscularly (IM) or intranasally(IN) with plasmid DNA containing the coding sequence of the MoPn MOMPgene (1095 bp), prepared as described in Example 1, or with the codingsequence of the C. trachomatis serovar L₂ CTP synthetase gene (1619 bp(refs. 10, 12), prepared by a procedure analogous described inExample 1. CTP synthetase is a conserved chlamydial cytoplasmic enzymecatalizing the final step in pyrimidine biosynthesis and is not known toinduce protective immunity. Negative control animals were injected withsaline or with the plasmid vector lacking an inserted chlamydial gene.

For IM immunization, both quardiceps were injected with 100 μg DNA in100 μl of saline per injection site on three occasions at 0, 3 and 6weeks. For IN immunization, anaesthetized mice aspirated 25 μl of salinecontaining 50 μg DNA on three occasions at 0, 3 and 6 weeks. As apositive control, a separate group of mice received 5×10⁶ inclusionforming units (IFUs) of MoPn EBs administered intraperitoneally inincomplete Freund's adjuvant according to the above schedule. At week 8,all groups of mice had sera collected for measuring antibodies and weretested for delayed-type hypersensitivity (DTH) to MoPn Ebs by footpadinjection (ref. 13).

A positive 48 and 72 hour DTH reaction was detected among mice immunizedwith MOMP DNA or with MoPn Ebs but not among mice immunized with theblank vector (see FIG. 1). The DTH reaction elicited with MOMP DNAdelivered intranasally was comparable to that observed among miceimmunized with EBs. No DTH reaction was detected among the groups ofmice vaccinated with CTP synthetase DNA (see Table 1 below). Thus,injection of MOMP DNA generated a DTH reaction that was capable ofrecall by naturally processed peptides from C. trachomatis EBs whileinjection of CTP synthetase DNA failed to do so.

EXAMPLE 3

This Example illustrates DNA immunization of mice and the generation ofantibodies.

Injection of CTP synthetase DNA as described in Example 2 resulted inthe production of serum antibodies to recombinant CTP synthetase(Table 1) (ref. 14). Antigen-specific serum Abs were measured by ELISA.Flat-bottom 96-well plates (Corning 25805, Corning Science Products,Corning, N.Y.) were coated with either recombinant chlamydialCTP-synthetase (1 μg/ml) or purified MoPn EBs (6×10⁴ IFU/well) overnightat 4° C. The Plates were rinsed with distilled water and blocked with 4%BSA PBS-Tween and 1% low fat skim milk for 2 hours at room temperature.Dilutions of sera samples were performed in 96-well round bottom platesimmediately prior to application on the antigen coated plates. Theplates were incubated overnight at 4° C. and washed ten times.Biotinylated goat anti-mouse IgG1 or goat anti-mouse IgG2a (SouthernBiotechnology Associates, Inc. Birmingham, Ala.) were next applied for 1hour at 37° C. After washing, streptoavidin-alkaline phosphataseconjugate (Jackson ImmunoResearch Laboratories, Inc. Mississagua,Ontario, Canada) were added and incubated at 37° C. for 30 min.Following another wash step, phosphatase substrate in phosphatase buffer(pH 9.8) was added and allowed to develop for 1 hour. Th plates wereread at 405 nm on a BIORAD 3550 microplate reader.

1gG2a antibody titers were approximately 10-fold higher than 1gG1antibody titers suggesting that DNA immunization elicited a moredominant T_(H1)-like response. Injection of MOMP DNA as described inExample 2 resulted in the production of serum antibodies to MOMP (Table2) as detected in an immunoblot assay (FIG. 2). However, neither CTPsynthetase DNA nor MOMP DNA immunized mice produced antibodies thatbound to native C. trachomatis EBs (Table 1), suggesting that theantibody responses may not to be the dominantly protective mechanism. Acomparison of serum 1gG subclasses, 1gG2a (Panels A and C and 1gG₁(Panels B and D) against MOMP protein (Panels A and B) or MoPn (Panels Cand D) induced by DNA immunization as described above, is contained inFIG. 4.

EXAMPLE 4

This Example illustrates DNA immunization of mice to achieve protection.

To investigate whether a cell-mediated immune response elicited by MOMPDNA was functionally significant, in vivo protective efficacy wasevaluated in mice challenged intranasally with 1×10³ IFU of C.trachomatis MoPn. To provide a measure of Chlamydia-induced morbidity,the loss in body weight was measured over 10 days following challengewith C. trachomatis (see FIG. 2, Panel A). Mice injected with theunmodified vector were used as negative controls and mice immunized withEBs were used as positive controls. Mice immunized with MOMP DNAintranasally maintained a body weight comparable to that observed amongEB immunized mice. Mice intramuscularly immunized with MOMP DNA lostbody mass but did so at a rate less than the negative control group.

A more direct measure of the effectiveness of DNA vaccination is theability of mice immunized with MOMP DNA to limit the in vivo growth ofChlamydia following a sublethal lung infection. Day 10 post-challenge isthe time of peak growth (ref. 13) and was chosen for comparison of lungtiters among the various groups of mice. Mice intranasally immunizedwith MOMP DNA had chlamydial lung titers that were over 1000-fold lower(log₁₀ IFU 1.3±0.3; mean±SEM ) than those of control mice immunized withthe blank vector (log₁₀ IFU 5.0±0.3; p<0.01) (see FIG. 2, Panel B). Miceintramuscularly immunized with MOMP DNA had chlamydial lung titers thatwere more than 10-fold lower than the unmodified vector group (p=0.01).Mice intranasally immunized with MOMP DNA had significantly lowerchlamydial lung titers than mice immunized with MOMP DNA intramuscularly(log₁₀ IFU 1.3±0.8 versus log₁₀ IFU 0.66±0.3 respectively; p=0.38). Thesubstantial difference (2.4 logs) in chlamydial lung titers observedbetween the intranasally and intramuscularly MOMP DNA immunized micesuggests that mucosal immunization is more efficient at inducing immuneresponses to accelerate chlamydial clearance in the lung. The lack ofprotective effect with the unmodified vector control confirms that DNAper se was not responsible for the immune response. Moreover, theabsence of protective immunity following immunization with CTPsynthetase DNA confirms that the immunity was specific to the MOMP DNA(see Table 1). FIG. 5 shows similar challenge data at a higher challengedose.

EXAMPLE 5

This Example describes the construction of p½MOMP.

A PCR cloned MoPn gene was constructed containing a deletion mutation incodon 177. This recitation yields a truncated MOMP protein containingapproximately 183 amino-terminal amino acids (ref. 10). This construct,termed p½MOMP, was cloned into the vector pcDNA3 (Invitrogen), in themanner described in Example 1.

EXAMPLE 6

This Example illustrates immunization of mice with P½MOMP.

Balb/c mice were immunized in the quadriceps three times at three weekintervals with 100 μg of p½MOMP DNA.

Fifteen days after the last immunization and 60 days after the firstinjection, mice were bled for measurement of serum antibodies of MoPnEBs in an EIA assay and were injected in the footpad with 25 μl (5×10⁴inclusion forming units) of heat killed EBs for measurement of DTH whichwas measured at 72 hours (ref. 13). Mice were intranasally challengedwith 1000 infectious units of MoPn and their body weight measured dailyfor the subsequent 10 days. At that time, mice were sacrificed andquantitative cultures of MoPn in the lung determined (ref. 13).

Table 3 shows that p½MOMP immunization elicited a positive DTH responseto footpad injection of MoPn EBs. Low titers (approximate titer 1/100)serum antibodies to surface determinants on EBs were also detected atday 60 post vaccination. Immunization with the unmodified vectorelicited neither serum antibodies nor a DTH response. FIG. 6, Panel Ashows that p½MOMP immunization evoked a protective immune response toMoPn challenge as measured by change in body weight post infection andby the in vivo growth of MoPn in lung tissue day 10 post challenge. Thein vivo growth among saline treated mice was log₁₀ 5.8±0.21 and amongp½MOMP immunized mice was log₁₀ 3.9±0.25, p<0.001, FIG. 2, Panel B. As apositive control, mice immunized with heat killed MoPn EBs or recoveredfrom prior infection with MoPn were markedly and equivalently protectedagainst challenged infection (p<0.0001).

As may be seen in this Example, using a frame-shift deletion mutant atcodon 177 of the MOMP gene, significant protective immunity to challengeinfection was elicited suggesting that protective sites can be found inthe amino terminal half of the protein.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a methodof nucleic acid, including DNA, immunization of a host, includinghumans, against disease caused by infection by strain of Chliamydia,specifically C. trachomatis, employing a non-replicating vector,specifically a plasmid vector, containing a nucleotide sequence encodinga major outer membrane protein (MOMP) of a strain of Chlamydia and apromoter to effect expression of MOMP in the host. Modifications arepossible within the scope of this invention.

TABLE 1 Serum antibody titers and delayed-type hypersensitivity (DTH)responses and in vivo growth of Chlamydia trachomatis following pCTPsynthetase or MoPn EB immunization. Results are presented as means ±SEM. anti-MoPn EB anti-rCTP synthetase log₁₀ IFU/lung antibodies (log₁₀)antibodies (log₁₀) anti-EB DTH d10 post IgG1 IgG2a IgG1 IgG2a (mm × 10²)challenge Saline (n = 9) <2 <2 <2 <2 4.5 ± 1.5 4.9 ± .24 pCTP synthetase<2 <2 3.8 ± .3 4.7 ± .1 1.4 ± 1.5 4.7 ± .13 (n = 11) EB (n = 4) 5.0 ± .24.8 ± .3 3.6 ± .8 2.9 ± 0  15.2 ± 2.0  0

TABLE 2 Serum antibody Elisa titers to Chlamydia trachomatis mousepneumonitis recombinant MOMP and Ebs were measured 60 days after theinitial immunization among mice immunized with blank vector alone(pcDNA3), vector containing the MOMP gene (pMOMP) and vector containingthe CTP synthetase gene (pCTP). Non-immunized mice were also tested.rMOMP EB IgG2a IgG1 IgG2a IgG1 pcDNA3 <2.6* <2.6 <2.6 <2.6 pMOMP 3.77 ±0.1 2.90 ± 0.14 3.35 ± 0.11 <2.6 pCTP ND ND <2.6 <2.6 Preimmunization<2.6 <2.6 <2.6 <2.6 *log₁₀ mean ± SE IgG isotype specific antibody titerND = not done

TABLE 3 Immune responses at day 60 following p½MOMP or EB immunization.EB IgG_(2a) DTH response antibody titer to EB Immunogen (log₁₀) (mm ×10²) EB (n = 13) 5.6 ± 0.4 9.6 ± 2.0 p½MOMP (n = 13) 2.0 ± 0     6 ± 1.6pcDNA3 (n = 13) 1.3 ± 0   1 ± 1

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2 1 35 DNA Chlamydia trachomatis 1 ggggatccgc caccatgctg cctgtggggaatcct 35 2 28 DNA Chlamydia trachomatis 2 ggggctcgag ctattaacgg aactgagc28

I claim:
 1. An immunogenic composition for intranasal or intramuscularadministration to a host for the generation in the host of a protectiveimmune response to a major outer membrane protein (MOMP) of a strain ofChlamydia trachornatis or Chlamydia pneumoniae, comprising anon-replicating vector suitable for DNA vaccine use, comprising: anucleotide sequence encoding said MOMP or an N-terminal fragment ofapproximately half full-length MOMP, and a cyomegalovirus promotersequence operatively coupled to said nucleotide sequence for expressionof said MOMP in the host; and a pharmaceutically-acceptable carriertherefor.
 2. The immunogenic composition of claim 1 wherein saidnucleotide sequence encodes full-length MOMP.
 3. The immunogeniccomposition of claim 1 wherein said strain of Chlamydia is a strain ofChlamydia trachomatis.
 4. The immunogenic composition of claim 3 whereinsaid non-replicating vector comprises plasmid pcDNA3 containing saidpromoter sequence and into which said nucleotide sequence is inserted inoperative relation to said promoter sequence.
 5. The immunogeniccomposition of claim 1 wherein said immune response is predominantly acellular immune response.
 6. The immunogenic composition of claim 1wherein said nucleotide sequence encodes said MOMP which stimulates arecall immune response following exposure to wild-type Chlamydia.
 7. Amethod of immunizing a host against disease caused by infection with astrain of Chlamydia trachomatis or Chlamydia pneunioniae, whichcomprises administering to said host intranasally or intramuscularly aneffective amount of a non-replicating vector comprising: a nucleotidesequence encoding a major outer membrane protein (MOMP) of a strain ofChlamydia trachomatis or Chlamydia pneumoniae or an N-terminal fragmentof approximately half the full-length MOMP, and a promoter sequenceoperatively coupled to said nucleotide sequence for expression of saidMOMP in the host.
 8. The method of claim 7 wherein said nucleotidesequence encodes full-length MOMP.
 9. The method of claim 7 wherein saidnucleotide sequence encodes an N-terminal fragment of approximately halfof full length MOMP.
 10. The method of claim 7 wherein said promotersequence is a cytomegalovirus promoter.
 11. The method of claim 7wherein said strain of Chlamydia is a strain of Chlamydia trachomatis.12. The method of claim 7 wherein said non-replicating vector comprisesplasmid pcDNA3 containing said promoter into which said nucleotidesequence is inserted in operative relation to said promoter sequence.13. The method of claim 7 wherein said immune response is predominantlya cellular immune response.
 14. The method of claim 7 wherein saidnucleotide sequence encodes said MOMP which stimulates a recall immuneresponse following exposure to wild-type Chlamydia.
 15. The method ofclaim 7 wherein said non-replicating vector is administeredintranasally.
 16. A method of using a gene encoding a major outermembrane protein (MOMP) of a strain of Chlamydia trachomatis orChlamycha pneumoniae or an N-terminal fragment of approximately half ofthe full-length MOMP, which comprises: isolating said gene, operativelylinking said gene to at least one control sequence to produce anon-replicating vector, said control sequence directing expression ofsaid MOMP or fragment thereof when introduced into a host to produce animmune response to said MOMP or fragment thereof, and introducing saidvector into a host intranasally or intramuscularly.
 17. The method ofclaim 16 wherein said gene encoding MOMP encodes full length MOMP. 18.The method of claim 16 wherein said gene encoding MOMP encodes anN-terminal fragment of approximately half of full-length MOMP.
 19. Themethod of claim 16 wherein said control sequence is a cytomegaloviruspromoter.
 20. The method of claim 16 wherein said strain of Chlamydia isa strain of Chlamydia trachomatis.
 21. The method of claim 16 whereinsaid non-replicating vector comprises plasmid pcDNA3 containing saidcontrol sequence into which said gene encoding MOMP is inserted inoperative relation to said control sequence.
 22. The method of claim 16wherein said immune response is predominantly a cellular immuneresponse.
 23. The method of claim 16 wherein said gene encodes said MOMPwhich stimulates a recall immune response following exposure towild-type Chlamydia.
 24. The method of claim 16 wherein said vector isintroduced into said host intranasally.